Generally speaking, DNA or gene chips have being developed for detection of infectious pathogens, disease-causing genes, mutations, and expression levels of genes, etc. In some instances, the technology has also been adapted for diagnosis at hospitals and clinics. In other instances, various technologies have been consolidated and optimized for convenient use and for reliable decisions in connection with clinical diagnosis.
For detection of certain DNA sequence(s) in a biological sample with use of a DNA chip, the following steps are typically followed:
(1) secure tissues, cells and/or biological fluids,
(2) extraction of DNA or RNA,
(3) amplification of certain sequences or nucleic acid regions of interest, typically by polymerase chain reaction (PCR), with use of appropriate primers in the presence of deoxynucleotide triphosphates, one of which is labeled with fluorescent compound,
(4) purification of the amplified DNA,
(5) add the purified amplified DNA to a solid surface, such as a glass slide, containing attached short single stranded DNAs that act as capture probes by being complementary to the amplified DNA,
(6) hybridization of the fluorescently labeled amplified DNA to a capture probe to form double stranded DNA attached to the solid surface,
(7) scanning the fluorescence of the double-stranded DNA formed on the surface of the chip, and
(8) analysis of the data.
These processes are long, and cumbersome, and loss of samples occur during purification of reaction products. The losses cause variation of the final results.
The Polymerase Chain Reaction (PCR) is used widely for amplification of a minute amount of a defined DNA sequence to be an amount sufficient for analysis such as DNA sequencing, detection by gel electrophoresis, and for DNA chip-based analysis. Generally, the PCR process involves denaturation of DNA (strand separation) at a high temperature, annealing of primers that are complementary to either ends of a DNA region to be amplified, and chain elongation from the annealed primers in the presence of thermostable DNA polymerase and the four deoxynucleotide triphosphates. These processes require specific temperatures: for example, 90° C. or above for denaturation, 55-65° C. for annealing, and 72° C. for chain elongation. Denaturation, annealing and chain elongation are repeated continuously until desired amount of DNA is amplified, typically 20-30 cycles. Therefore, PCR requires a programmable thermocycler.
A DNA chip is typically a slide on which capture probes (short single-stranded DNA) are immobilized in a high density format. They are used for analysis of the presence of certain DNA or RNA sequences, the presence of mutation(s), and expression (transcription) of certain genes in cells or tissues.
Usually, a glass slide is coated with chemicals to attach DNA to the glass. The chemicals should not only attach the DNA to the glass surface but also minimize non-specific binding and noise. For example, the chemicals contain silanated, silylated, or poly-L-lysine as a source of amine or aldehyde group for attachment of DNA. Recently, dendrons have been developed for use in biochip. When a chip is coated with dendrons, one can control spacing between capture probes and this also allows reduction of steric hinderance and concomitant increase in sensitivity. Korean patent 10-0383080-0000 and published U.S. Patent Application 2005/0037413 describe dendrons provide controlled spacing as well as density of amines on dendron.
When DNA chip slides are used, the sequence of steps is to amplify the DNA (labeling the DNA with fluorescent group at the same time) and then purify the DNA. The amplified DNA is denatured by heating and added to the surface of capture probes for annealing. After hybridization, the slide is washed and the fluorescent double-stranded DNA is detected by scanning and the data are analyzed.
As described above, the use of DNA chip requires extraction of DNA or RNA from cells, gene amplification and purification, and DNA hybridization. This requires different instruments for gene amplification and hybridization, and considerable time is required for gene amplification and purification (as much as one day). Also, the cost of chemicals and disposable items is high. There is also loss of sample during purification of gene amplification products. Therefore, there is need for streamlining the cumbersome processes involved in use of gene chips.
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